Oscarliu117 (Talk | contribs) |
Oscarliu117 (Talk | contribs) |
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<li>Leave on immediately on ice for 30 minutes.</li> | <li>Leave on immediately on ice for 30 minutes.</li> | ||
<li>Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with | <li>Aliquot 100 μl into 1.5 ml centrifuge tubes and snap freeze immediately with | ||
− | + | liquid nitrogen.</li> | |
<li>Store the frozen cells in the -80℃ freezer.</li> | <li>Store the frozen cells in the -80℃ freezer.</li> | ||
</ol> | </ol> | ||
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<div class="collapse multi-collapse" id="Plasmid_Extraction"> | <div class="collapse multi-collapse" id="Plasmid_Extraction"> | ||
<p class="pcontent"></br></br> | <p class="pcontent"></br></br> | ||
− | + | <ol> | |
− | + | <li>Transfer 1.4 ml of well-grown bacterial culture to | |
− | + | a centrifuge tube.</li> | |
− | + | <li>Centrifuge the tube at 16,000 xg for 1 minute to pellet the cells and | |
− | + | discard the supernatant completely.</li> | |
− | + | <li>Add 200 µl of FAPD1 Buffer (RNaseA added) to the cell pellet and resuspend | |
− | + | the cells completely by pipetting.</li> | |
− | + | ||
− | + | <ul> | |
− | + | <li>Make sure that RNaseA has been added into FAPD1 Buffer when first use.</li> | |
− | + | <li>No cell pellet should be visible after resuspension of the cells.</li> | |
− | + | ||
− | + | </ul> | |
− | + | ||
− | + | <li>Add 200 µl of FAPD2 Buffer and gently invert the tube 5 ~ 10 times. | |
− | + | Incubate | |
− | + | the sample mixture at room temperature for 2 ~ 5 minutes to lyse the cells.</li> | |
− | + | ||
− | + | <ul> | |
− | + | <li>Do not vortex, vortex may shear genomic DNA. If necessary, continue | |
− | + | inverting the tube until the lysate become clear.</li> | |
− | + | <li>Make sure the tube transfer to clarify from turbid.</li> | |
− | + | <li>Do not proceed with the incubation over 5 minutes.</li> | |
− | + | </ul> | |
− | + | ||
− | + | <li>Add 300 µl of FAPD3 Buffer and invert the tube 5 ~ 10 times immediately to | |
− | + | neutralize the lysate.</li> | |
− | + | ||
− | + | <ul> | |
− | + | <li>Invert immediately after adding FAPD3 Buffer will avoid asymmetric | |
− | + | precipitation.</li>Invert immediately after adding FAPD3 Buffer will | |
− | + | avoid asymmetric precipitation. | |
− | + | </ul> | |
− | + | ||
− | + | <li>Centrifuge at 16,000 xg for 3 minutess. to clarify the lysate. During | |
− | + | centrifugation, place a FAPD Column in a Collection Tube.</li> | |
− | + | <li>Transfer the supernatant carefully to the FAPD Column and centrifuge at | |
− | + | 16,000 xg for 1 minute. Discard the flow-through and place the column back | |
− | + | to | |
− | + | the Collection Tube.</li> | |
− | + | ||
− | + | <ul> | |
− | + | <li>Do not transfer any white pellet into the column.</li> | |
− | + | </ul> | |
+ | |||
+ | <li>Add 400 µl of W1 Buffer to the FAPD Column and centrifuge at 16,000 xg for | ||
+ | 1 | ||
+ | minute. Discard the flow-through and place the column back to the | ||
+ | Collection | ||
+ | Tube.</li> | ||
+ | <li>Add 600 µl of Wash Buffer to the FAPD Column and centrifuge at 16,000 xg | ||
+ | for | ||
+ | 1 minute. Discard the flow-through and place the column back to the | ||
+ | Collection | ||
+ | Tube.</li> | ||
+ | |||
+ | <ul> | ||
+ | <li>Make sure that ethanol (96 ~ 100 %) has been added into Wash Buffer | ||
+ | when | ||
+ | first use.</li> | ||
+ | </ul> | ||
+ | |||
+ | <li>Centrifuge at 16,000 xg for an additional 3 minutes to dry the FAPD | ||
+ | Column.</li> | ||
+ | |||
+ | <ul> | ||
+ | <li>Important step! The residual liquid should be removed thoroughly on | ||
+ | this | ||
+ | step.</li> | ||
+ | </ul> | ||
+ | |||
+ | <li>Place the FAPD Column to a new 1.5 ml microcentrifuge tube.</li> | ||
+ | <li>Add 30 µl of Elution Buffer or ddH2O to the membrane center of the FAPD | ||
+ | Column. Stand the column for 3 minute.</li> | ||
+ | <ul> | ||
+ | <li>Important step! For effective elution, make sure that the elution | ||
+ | solution is | ||
+ | dispensed on the membrane center and is absorbed completely.</li> | ||
+ | <li>Do not elute the DNA using less than suggested volume (50 µl). It will | ||
+ | lower the final yield.</li> | ||
+ | </ul> | ||
+ | |||
+ | <li>Centrifuge at 16,000 xg for 3 minute to elute plasmid DNA and store the DNA | ||
+ | at -20 ℃.</li> | ||
+ | </ol> | ||
+ | </p> | ||
Revision as of 12:04, 29 September 2018